Blocking mGLuR5-mediated neuronal vulnerability to Aβ and α-syn in the hippocampal neurons via lentivirus shRNA delivery in vitro. A. Confocal imaging and B Western blot of primary neuronal hippocampal cells, which endogenously express mGluR5. LV-sh-luciferase (control LV-sh) had no effect on the expression of mGluR5, while LV-sh-mGluR5 effectively reduced endogenous mGluR5 expression by 80%. C. Confocal microscopy of neuronal cells co-infected with LV-control or LV-α-syn in the presence of either LV-sh-Luc or LV-sh-mGluR5 and treated with vehicle or Aβ oligomers and double labeled with antibodies against α-syn and MAP 2. D. Image analysis revealed that neurite distance (MAP 2) was reduced 35-40% in neuronal cells infected with LV-α-syn, and when this system was challenged with Aβ oligomers, neurite length was further reduced to 40-50% of control. While LV-sh-Luc had no effect on neurite length in either the presence or absence of Aβ oligomers, down modulation of mGluR5 using LV-sh-mGluR5 rescued neurite length in neuronal cells treated with LV-α-syn, as well as with the combined toxicity of LV-α-syn and Aβ oligomers. E. Image analysis of α-syn revealed that neuronal cells infected with LV-α-syn expressed higher levels of α-syn compared to untreated cells, and that Aβ oligomers further increased this expression compared with cells only treated with LV-α-syn. Down modulation of mGluR5 with LV-sh-mGluR5 had no effect on α-syn levels. F. Neuronal survival by LDH, showing that down-modulation of mGluR5 is protective form the toxic effects of Aβ oligomers and α-syn. * = p-value < 0.05 by one-way ANOVA and Dunnett’s post hoc analysis compared to vehicle-treated uninfected neurons. # = p < 0.05 by one-way ANOVA and Tukey-Kramer post hoc analysis when compared to LV-α-syn + Aβ oligomers.